Studies on the factors affecting gene expression in yeast.

酵母基因表达影响因素的研究。

基本信息

批准号：
61580225
负责人：
KIKUCHI Yoshiko
金额：
$ 1.02万
依托单位：
Toho University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

Many factors should be involved in gene expression in yeast. In this project, we focused on the effiect of the stability of vectore. We have isolated two types of nuclear mutants of Saccharomyces cerevisiae, which affect the maintenance of plasmid and mini-chromosomes.1. map1; We have isolated host mutants in which the 2<micro>m plasmid is poorly maintained. All the mutants tested constituted one complementation group, which was designated map1. Minichromosomes carrying chromosomal replication oringin and a centromere were also affected in the mutants. Two types of hybrid plasmids generated in vivo and in vitro appeared to compensate for the mutations and had DNA regions containing multiple ARS ot a set of 2<micro>m inverted repeat sequences. These results suggested that poor maintenance of plasmids was due to low levels of replication, probably at the initiation of replication.2. gst1; A new temperature-sensitive mutant, gst1 was isolated. At non-permissive temperature the mutant cells with large buds accumulated and DNA synthesis was substantially arrested. From the reciprocal experiment of temperature-shift ands mating factor treatment, it was shown that the execution point was post'START'. This suggested that the mutation affected the G1-to-S phase transition in the cell cycle. A DNA clone complementing the gst-1 mutation was isolated from gene library, and gst1 was mapped in chr 4R, by Southern blotting of cloned sequence to the individual yeast chromosome DNA by OFAGE system and by genetic analysis. The gene product was tentatively assigned from DNA sequencing analysis, as a protein of MW 76565 which contained consensus sequences for a target site of cAMP-dependent protein kinase and for GTPase with extensive homology to polypeptide chain elongation factor EF1<alpha>.

酵母中的基因表达应涉及许多因素。在这个项目中，我们专注于矢量稳定性的有效性。我们已经分离了酿酒酵母的两种类型的核突变体，这会影响质粒和迷你染色体的维持。1。 MAP1;我们有分离的宿主突变体，其中2 <micro> m质粒的维持不足。所有测试的突变体构成一个互补组，该组被指定为MAP1。携带染色体复制oringin和Cencermere的微型染色体在突变体中也受到影响。在体内产生和体外产生的两种类型的杂化质粒似乎可以补偿突变，并且DNA区域包含多个ARS OT一组2 <micro> m倒置重复序列。这些结果表明，质粒的维持不力是由于复制水平较低，可能是在复制的开始时。2。 GST1;一个新的温度敏感突变体GST1分离出来。在非耐药温度下，积累了大芽和DNA合成的突变细胞被实质上捕获。从温度转移和交配因子处理的相互实验中，可以证明执行点是“开始后”。这表明该突变影响了细胞周期中的G1至S相变。从基因文库中分离出与GST-1突变相互补充的DNA克隆，并通过Of ofage System和遗传分析将克隆序列与单个酵母染色体DNA的Southern斑点映射为CHR 4R。基因产物是从DNA测序分析中分配的，该分析是MW 76565的蛋白质，其中包含了cAMP依赖性蛋白激酶目标位点的共识序列和具有与多肽链链伸长因子EF1 <alpha>的GTPase具有广泛同源性的GTPase。